- Email: [email protected]
93 Loss of complex I due to mitochondrial DNA mutations in renal oncocytoma
Abstracts / Mitochondrion 7 (2007) 404–433 ined mitochondrial effects of several families of drugs used for treatment of diabetes and hyperlipidemia, including thiazolidinediones, fibrates, and statins. Respiration of isolated rat liver mitochondria during State 2 (oxidizable substrate) and State 3 (substrate plus ADP) was monitored using a fluorescent probe that reports PO2. Of the six thiazolidinedioness examined, ciglitazone, troglitazone and darglitazone potently uncoupled State 2 respiration, and also inhibited State 3 respiration. Within the fibrates, gemfibrozil was the most potent uncoupler of State 2, followed by fenofibrate, while gemfibrozil also inhibited State 3 respiration. Of the six statins evaluated, simvastatin, fluvastatin, and lovastatin impaired mitochondrial respiration the most, whereas pravastatin and atorvastatin showed only modest effects. The statins are bio-accumulated into fasttwitch muscle fibers by monocarboxylate transporter isoform 4, which explains not only why this fiber type selectively succumbs during rhabdomyolysis, but also why cardiac muscle is spared. Our findings indicate that some members of each class impair mitochondrial function, and the rankorder of potency correlates with clinical adverse events. We are now investigating a wider range of pharmaceuticals, but it is increasingly clear that pre-clinical assessments of drug-induced mitochondrial impairment can help drive drug development and thus improve drug safety. doi:10.1016/j.mito.2007.08.095
92 Normal biochemical analysis of the oxidative phosphorylation (OXPHOS) system in a child with POLG mutations: A cautionary note! Maaike C. de Vries a,*, Eva Morava a, Richard J. Rodenburg a, Christoph Korenke b, Lambert P.W. van den Heuvel a, Jan A.M. Smeitink a a Nijmegen Centre for Mitochondrial Disorders at the Departments of Pediatrics and Laboratory of Pediatrics and Neurology, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands; b Klinikum Oldenburg, Department of Pediatrics, Oldenburg, Germany In childhood, mutations in the polymerase gamma (POLG1) gene have so far been described in patients severe combined oxidative phosphorylation (OXPHOS) deficiencies and originally in patients with Alpers syndrome. Here we report on a 7-year-old boy carrying POLG1 mutations, but with normal oxidative phosphorylation analysis in muscle, fibroblasts and liver. He presented during the first year of life with severe psychomotor developmental delay, hypotonia and ataxia together with visual impairment and sensori-neural deafness. At the age of three years old the child developed epilepsy with periods of status epilepticus partialis. Laboratory investigations showed normal liver functions, normal lactic acid concentrations in blood, but elevated lactic acid in cerebrospinal fluid. Organic acids and amino acids analysis showed normal values. No abnormalities were found by imaging of the brain by MRI and MRS. Measurements of the oxidative phosphorylation system were performed in a frozen muscle biopsy and showed no deficiencies of the enzyme complexes. Because of a strong suspicion of a mitochondrial disorder the biochemical analysis of the OXPHOS system was repeated in a fresh muscle biopsy, liver biopsy and fibroblasts. In muscle, the ATP production from pyruvate was normal in combination with normal substrate oxidation rates and normal enzyme complex activities. In liver and fibroblasts, normal activities of the enzyme complexes were found. Histological analysis of the biopsies showed no abnormalities, except from some cholestasis in the hepatocytes. Mutation analysis of the POLG1 gene showed combined heterozygosity with two known mutations, the A467T and G848S mutation. The normal findings by biochemical analysis of the OXPHOS system in our patient show the complexity and heterogeneity of the population of patients with POLG1 mutations in childhood and warrants, based on the high frequency of POLG mutations in childhood, a liberal strategy with respect to mutation analysis. doi:10.1016/j.mito.2007.08.096
431
93 Loss of complex I due to mitochondrial DNA mutations in renal oncocytoma Olaf Stanger a, Edith Mu¨ller b, Franz Zimmermann b, Martina Wiesbauer c, Johannes A. Mayr b, Bernhard Paulweber d, Bernhard Iglseder e, Wilfried Renner f, Waltraud Eder b, Barbara Kofler g,* a Department of Biological Chemistry, Biocentre, Innsbruck Medical University, Innsbruck, Austria; b Department of Cellular Biology, University of Salzburg, Austria; c Molecular Biology, University of Salzburg, Austria; d Department of Cardiology, Paracelus Private Medical University, University of Salzburg, Austria; e Paracelus Private Medical University, University of Salzburg, Austria; f Department of Opthalmology, Medical University of Graz, Austria; g Clinical Institute of Medical and Chemical Laboratory Diagnostics, University and General Hospital, Medical University, Auenbruggerplatz 30, 8036 Graz, Austria Many solid tumors exhibit abnormal aerobic metabolism characterized by increased glycolytic capacity and decreased cellular respiration. Recently, mutations in the mitochondrial enzymes fumarate hydratase and succinate dehydrogenase have been identified in certain tumor types, thus demonstrating a direct link between mitochondrial energy metabolism and tumorigenesis. Renal oncocytomas are benign tumors accounting for approximately 5% of all renal cell neoplasias. A characteristic feature of oncocytomas is the accumulation of a large number of mitochondria. In order to elucidate the cause of the mitochondrial alterations in oncocytomas, we investigated the activities of respiratory chain enzymes and screened for mtDNA mutations in renal oncocytomas. Here we show that loss of respiratory chain complex I (NADH:ubiquinone oxidoreductase) is associated with renal oncocytoma. Enzymatic activity of complex I was undetectable or greatly reduced in the tumor samples (n = 13). Blue Native gel electrophoresis of the multi-subunit enzyme complex revealed a lack of assembled complex I in most of the renal oncocytomas, and one sample showed an incompletely assembled enzyme. Mutation analysis of the mitochondrial DNA (mtDNA) showed frame-shift mutations in the genes encoding the ND1, ND4 and ND5 subunits of complex I in eight of the 13 tumors. One oncocytoma had an A3243G mutation in the mitochondrial tRNALeu(UUR) gene, which is a frequent cause of the MELAS (mitochondrial myopathy, encephalomyopathy, lactic acidosis, and stroke-like episodes) syndrome. Our findings reveal for the first time that complex I can be regarded as a mitochondrial tumor suppressor, at least in the case of renal oncocytomas. Our results also support the hypothesis of Otto Warburg, proclaimed more than 80 years ago, that irreversible damage to aerobic cellular respiration is a primary event in tumor development. doi:10.1016/j.mito.2007.08.097
94 Mitochondrial lipid and electron transport chain abnormalities in mouse brain tumors Michael A. Kiebish *, Xianlin Han, Hua Cheng, Thomas N. Seyfried Department of Internal Medicine, Washington University School of Medicine, St. Louis, MO 63110, USA Defects in mitochondrial metabolism have long been considered a hallmark of cancer. Mitochondria can be altered at the protein and mtDNA level, yet little attention has focused on the effects of alterations to mitochondrial lipids. Mitochondrial lipids play a crucial role in regulating enzyme activity, maintaining membrane fluidity, and contributing to the proton gradient. We analyzed the lipid composition of mitochondria isolated from transformed astrocytes and a series of murine brain tumors, including CT-2A, EPEN, VM-NM1, VM-M2, and VM-M3 grown in vitro. Purity of isolated mitochondria was accessed by Western blot, using markers for enrichment (Complex 4 and MAO-A) and contamination (b-tubulin, PCNA, Tuberin, and Calnexin).
92 Normal biochemical analysis of the oxidative phosphorylation (OXPHOS) system in a child with POLG mutations: A cautionary note! Maaike C. de Vries a,*, Eva Morava a, Richard J. Rodenburg a, Christoph Korenke b, Lambert P.W. van den Heuvel a, Jan A.M. Smeitink a a Nijmegen Centre for Mitochondrial Disorders at the Departments of Pediatrics and Laboratory of Pediatrics and Neurology, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands; b Klinikum Oldenburg, Department of Pediatrics, Oldenburg, Germany In childhood, mutations in the polymerase gamma (POLG1) gene have so far been described in patients severe combined oxidative phosphorylation (OXPHOS) deficiencies and originally in patients with Alpers syndrome. Here we report on a 7-year-old boy carrying POLG1 mutations, but with normal oxidative phosphorylation analysis in muscle, fibroblasts and liver. He presented during the first year of life with severe psychomotor developmental delay, hypotonia and ataxia together with visual impairment and sensori-neural deafness. At the age of three years old the child developed epilepsy with periods of status epilepticus partialis. Laboratory investigations showed normal liver functions, normal lactic acid concentrations in blood, but elevated lactic acid in cerebrospinal fluid. Organic acids and amino acids analysis showed normal values. No abnormalities were found by imaging of the brain by MRI and MRS. Measurements of the oxidative phosphorylation system were performed in a frozen muscle biopsy and showed no deficiencies of the enzyme complexes. Because of a strong suspicion of a mitochondrial disorder the biochemical analysis of the OXPHOS system was repeated in a fresh muscle biopsy, liver biopsy and fibroblasts. In muscle, the ATP production from pyruvate was normal in combination with normal substrate oxidation rates and normal enzyme complex activities. In liver and fibroblasts, normal activities of the enzyme complexes were found. Histological analysis of the biopsies showed no abnormalities, except from some cholestasis in the hepatocytes. Mutation analysis of the POLG1 gene showed combined heterozygosity with two known mutations, the A467T and G848S mutation. The normal findings by biochemical analysis of the OXPHOS system in our patient show the complexity and heterogeneity of the population of patients with POLG1 mutations in childhood and warrants, based on the high frequency of POLG mutations in childhood, a liberal strategy with respect to mutation analysis. doi:10.1016/j.mito.2007.08.096
431
93 Loss of complex I due to mitochondrial DNA mutations in renal oncocytoma Olaf Stanger a, Edith Mu¨ller b, Franz Zimmermann b, Martina Wiesbauer c, Johannes A. Mayr b, Bernhard Paulweber d, Bernhard Iglseder e, Wilfried Renner f, Waltraud Eder b, Barbara Kofler g,* a Department of Biological Chemistry, Biocentre, Innsbruck Medical University, Innsbruck, Austria; b Department of Cellular Biology, University of Salzburg, Austria; c Molecular Biology, University of Salzburg, Austria; d Department of Cardiology, Paracelus Private Medical University, University of Salzburg, Austria; e Paracelus Private Medical University, University of Salzburg, Austria; f Department of Opthalmology, Medical University of Graz, Austria; g Clinical Institute of Medical and Chemical Laboratory Diagnostics, University and General Hospital, Medical University, Auenbruggerplatz 30, 8036 Graz, Austria Many solid tumors exhibit abnormal aerobic metabolism characterized by increased glycolytic capacity and decreased cellular respiration. Recently, mutations in the mitochondrial enzymes fumarate hydratase and succinate dehydrogenase have been identified in certain tumor types, thus demonstrating a direct link between mitochondrial energy metabolism and tumorigenesis. Renal oncocytomas are benign tumors accounting for approximately 5% of all renal cell neoplasias. A characteristic feature of oncocytomas is the accumulation of a large number of mitochondria. In order to elucidate the cause of the mitochondrial alterations in oncocytomas, we investigated the activities of respiratory chain enzymes and screened for mtDNA mutations in renal oncocytomas. Here we show that loss of respiratory chain complex I (NADH:ubiquinone oxidoreductase) is associated with renal oncocytoma. Enzymatic activity of complex I was undetectable or greatly reduced in the tumor samples (n = 13). Blue Native gel electrophoresis of the multi-subunit enzyme complex revealed a lack of assembled complex I in most of the renal oncocytomas, and one sample showed an incompletely assembled enzyme. Mutation analysis of the mitochondrial DNA (mtDNA) showed frame-shift mutations in the genes encoding the ND1, ND4 and ND5 subunits of complex I in eight of the 13 tumors. One oncocytoma had an A3243G mutation in the mitochondrial tRNALeu(UUR) gene, which is a frequent cause of the MELAS (mitochondrial myopathy, encephalomyopathy, lactic acidosis, and stroke-like episodes) syndrome. Our findings reveal for the first time that complex I can be regarded as a mitochondrial tumor suppressor, at least in the case of renal oncocytomas. Our results also support the hypothesis of Otto Warburg, proclaimed more than 80 years ago, that irreversible damage to aerobic cellular respiration is a primary event in tumor development. doi:10.1016/j.mito.2007.08.097
94 Mitochondrial lipid and electron transport chain abnormalities in mouse brain tumors Michael A. Kiebish *, Xianlin Han, Hua Cheng, Thomas N. Seyfried Department of Internal Medicine, Washington University School of Medicine, St. Louis, MO 63110, USA Defects in mitochondrial metabolism have long been considered a hallmark of cancer. Mitochondria can be altered at the protein and mtDNA level, yet little attention has focused on the effects of alterations to mitochondrial lipids. Mitochondrial lipids play a crucial role in regulating enzyme activity, maintaining membrane fluidity, and contributing to the proton gradient. We analyzed the lipid composition of mitochondria isolated from transformed astrocytes and a series of murine brain tumors, including CT-2A, EPEN, VM-NM1, VM-M2, and VM-M3 grown in vitro. Purity of isolated mitochondria was accessed by Western blot, using markers for enrichment (Complex 4 and MAO-A) and contamination (b-tubulin, PCNA, Tuberin, and Calnexin).